Thirty-Seven Human Cases of Sparganosis from Ethiopia and South Sudan Caused by Spirometra Spp
Total Page：16
File Type：pdf, Size：1020Kb
Am. J. Trop. Med. Hyg., 93(2), 2015, pp. 350–355 doi:10.4269/ajtmh.15-0236 Copyright © 2015 by The American Society of Tropical Medicine and Hygiene Case Report: Thirty-Seven Human Cases of Sparganosis from Ethiopia and South Sudan Caused by Spirometra Spp. Mark L. Eberhard,* Elizabeth A. Thiele, Gole E. Yembo, Makoy S. Yibi, Vitaliano A. Cama, and Ernesto Ruiz-Tiben Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, Georgia; Ethiopia Dracunculiasis Eradication Program, Federal Ministry of Health, Addis Ababa, Ethiopia; South Sudan Guinea Worm Eradication Program, Ministry of Health, Juba, Republic of South Sudan; The Carter Center, Atlanta, Georgia Abstract. Thirty-seven unusual specimens, three from Ethiopia and 34 from South Sudan, were submitted since 2012 for further identification by the Ethiopian Dracunculiasis Eradication Program (EDEP) and the South Sudan Guinea Worm Eradication Program (SSGWEP), respectively. Although the majority of specimens emerged from sores or breaks in the skin, there was concern that they did not represent bona fide cases of Dracunculus medinensis and that they needed detailed examination and identification as provided by the World Health Organization Collaborating Center (WHO CC) at Centers for Disease Control and Prevention (CDC). All 37 specimens were identified on microscopic study as larval tapeworms of the spargana type, and DNA sequence analysis of seven confirmed the identification of Spirometra sp. Age of cases ranged between 7 and 70 years (mean 25 years); 21 (57%) patients were male and 16 were female. The presence of spargana in open skin lesions is somewhat atypical, but does confirm the fact that populations living in these remote areas are either ingesting infected copepods in unsafe drinking water or, more likely, eating poorly cooked paratenic hosts harboring the parasite. INTRODUCTION gated within 24 hours. Any emergent worms are collected and preserved in alcohol and sent to CDC for subsequent Sparganosis, or human infection with the larval plerocer- examination. Thirty-seven of those specimens, three from coid stage of members of the genus Spirometra (Cestoda: Ethiopia and 34 from South Sudan, were microscopically Diphyllobothriidea), is a rather common infection in south- 1–4 identified as spargana; 36 were collected through the national east (SE) Asia, but is much less frequently reported in programs to eliminate GWD and one from a surgical proce- other areas of the world, despite being a common infection dure for varicose vein. The three specimens submitted from in cats and dogs throughout much of the world. In Africa, Ethiopia over that time were out of a total of 21 specimens, there are limited reports of human cases dating back to 1907 and for South Sudan, the 34 spargana were out of a total of when the first case was noted, and since then there have 161 submitted samples. The cases ranged from 7 to 70 years been sporadic reports and records of human cases although of age, with a median age of 25 years. Twenty-one (57%) of these same authors have speculated that human sparganosis 5–7 the cases were male, 16 were female. For the 34 cases from is a relatively common infection, at least in east Africa. South Sudan, a map showing the location of those cases is As the end of the campaign to eradicate Guinea worm dis- provided (Figure 1). Those 34 cases emerged in 17 different ease (GWD) progresses and the number of endemic countries villages, and in those with multiple cases, the number of cases and cases decreases, there is increased effort to detect and per each individual village were 2, 2, 4, 5, and 9. Specimens contain each case. Concurrent with this is the need to con- were taken from various locations on the body (Figure 2), firm that each case is accurately identified as Dracunculus including thigh, lower leg, ankle, foot, knee, and penis and medinensis so that appropriate actions at the country pro- measured between 0.5 and 30 cm long, were whitish-tan in gram level can be undertaken. However, not all specimens 8,9 color, and often demonstrated a thicker anterior end and a recovered from or emerging via the skin are Dracunculus. very thin posterior end (Figure 3). Under low-power micro- Up to now, the most common non-Dracunculus recovered by scopic evaluation, rudimentary bothria (cephalic grooves) typical national programs and submitted to the World Health Organi- of pseudophyllidian tapeworms was clearly evident on some zation Collaborating Center (WHO CC) at Centers for Dis- specimens. However, not all specimens contained the anterior ease Control and Prevention (CDC) has been Onchocerca end, and ranged from short to very long ribbon-like pieces of volvulus. However, over the past 3 years, an increasing num- worm (Figure 4). In those specimens, identification was a bit ber of spargana have been submitted and this report briefly more challenging, but the presence of calcareous corpuscles details 37 such cases, two each from 2012 and 2013, and 33 (Figure 5) in the somatic matrix was particularly helpful in from 2014. establishing that the organism was a larval tapeworm. Nine of the submitted specimens were subjected to DNA CASE SERIES REPORT sequencing to further verify the morphological identification. In brief, whole genomic DNA was extracted using a Gentra As part of routine, ongoing case detection and containment Puregene Tissue Kit (Qiagen, Frederick, MD) and used for activities in remaining endemic countries, all suspect cases amplification of the nuclear 18S rDNA and mitochondrial (and rumors) of dracunculiasis are supposed to be investi- cytochrome oxidase 1 (CO1) genes. The 18S rDNA amplifica- tion was performed with general nematode primers commonly used in molecular verification of D. medinensis to confirm that *Address correspondence to Mark L. Eberhard, Division of 10 Parasitic Diseases and Malaria, Centers for Disease Control and samples were not morphologically atypical guinea worms. In Prevention, 1600 Clifton Road, Atlanta, GA 30333. E-mail: mle1@ all nine cases, 18S rDNA reactions were negative, supporting cdc.gov the original diagnosis that specimens were not D. medinensis. 350 HUMAN SPARGANOSIS IN EAST AFRICA 351 FIGURE 1. Map of South Sudan indicating the location of 17 villages or village clusters where 34 cases of human sparganosis were detected during 2013–2014. CO1 sequences were generated with a custom set of primers the 983 bp of shared CO1 sequence and were polymorphic at designed to serve as a general primer set for diphyllobothriid 10 single nucleotide sites (average pairwise polymorphism: CO1 amplification. The forward primer sCO1 F (5′-GAT 2.86 nucleotides, range: 0–8 nucleotides). BLAST queries of AGHMRGGGTGTTGATYTT-3′) and reverse primer sCO1 the National Center for Biotechnology Information (NCBI) R(5′-CCARATAGCATGATGCAAA-3′; modified from the nucleotide database indicated that, of available sequences, the diphyllobothriid Cox1Reverse primer of Wicht and others11) African spargana described here had highest similarity to were used for polymerase chain reaction (PCR) amplification. Spirometra erinaceieuropaei (89–90% identity over 983 bp). Sig- For cycle sequencing of PCR product, two internal primers nificant homologies were also returned for the diphyllobothriid (sCO1 intF: 5′-TGTTTACDGTRGGDTTRGAYG-3′ and genera Sparganum, Diphyllobothrium,andDiplogonoporus sCO1 intR: 5′-CAAGCRTAMCCBGACTCRT-3′)wereused and the cyclophyllid genus Echinococcus,with≥ 91% coverage in addition to the PCR primers so as to ensure complete of African spargana sequences. bidirectional coverage. Genetic distances (Kimura 2-parameter model, bootstrap Seven of the nine specimens were successfully amplified in N = 1,000) and phylogenetic trees (maximum-likelihood using 50 μL reactions comprising 1 μL of whole genomic DNA, 1X a GTR+G+I substitution model) were calculated and inferred, Platinum® Blue PCR Supermix (Invitrogen, New York, NY), respectively, in MEGA v6.012 (freely available at http://www and 0.4 μM of each CO1 primer. Cleaned PCR product .megasoftware.net) to further investigate the taxonomic relation- (StrataPrep PCR Purification Kit [Agilent Technologies, Foster ship among the African spargana, Spirometra erinaceieuropaei, City, CA]) was sequenced using the BigDye® Terminator v3.1 and other diphyllobothriid and Echinococcus species known or cycle sequencing kit (Applied Biosystems, New York, NY) previously reported to occur in humans. Representative CO1 andanalyzedona3130xl Genetic Analyzer (Applied Bio- gene sequences of Spirometra erinaceieuropaei (AB015754), systems). Electropherograms were visually inspected and the Sparganum proliferum (AB015753), Diphyllobothrium ditremum sequences trimmed and assembled with ChromasPro v1.7.4 (FM209182), D. nihonkaiense (AB268585), D. latum (AB269325), (Technelysium, South Brisbane, Australia). High-quality par- D. dendriticum (KC812048), Diplogonoporus balaenopterae tial CO1 gene sequences of 996–1,027 base pairs (bp) were (AB425839), Dg. grandis (AB425840), Echinococcus canadensis generated for the seven successfully amplified specimens and (AB745463), E. multilocularis (AB510023), and E. granulosus were submitted to GenBank (accession numbers KM248530- (GQ168812) were obtained from GenBank, with the cestodarian 248536). Overall, the seven specimens had 99% identity over species Gyrocotyle urna (JQ268546) serving as the out-group. 352 EBERHARD AND OTHERS FIGURE 2. Clinical presentation of a case of sparganosis illustrating the progression of the emergence